Regional differences in the inductive activity of the mesenchyme of the embryonic mouse urogenital sinus

Embryonic urogenital sinuses (UGS) of 16-day-old mice were divided into two or three zones (cranial half and caudal half; or cranial third, intermediate, and caudal thirds). Following tryptic digestion, these zones were separated into their mesenchymal (UGM) and epithelial (UGE) components. The cranial and caudal zones of the UGM were recombined separately with the different zones of the UGE or with adult mouse urinary bladder epithelium (BLE). Following four weeks of growth in syngeneic male hosts, tissue recombinants of cranial UGM + caudal UGE contained numerous prostatic ducts and urethral glands. Conversely, recombinants composed of caudal UGM + cranial UGE developed into fibromuscular tissue covered with urethral epithelium and did not contain prostate, but occasionally contained urethral glands. Urethral glands were usually present in grafts of cranial UGM + cranial UGE or in grafts of the intact intermediate third of the UGS. In heterotypic recombinants of the UGM + adult BLE, prostatic glands were induced only when cranial UGM was utilized. Urethral glands were not observed in tissue recombinants prepared with adult BLE. These data suggest that regional differences in mesenchymal inductive ability and epithelial responsiveness play a role in the harmonious morphogenesis of the male lower genitourinary tract.     

Stromal-epithelial interactions: II. Regulation of prostatic growth by embryonic urogenital sinus mesenchyme

Embryonic urogenital sinus mesenchyme (UGM) has been demonstrated previously to be a potent inductor of prostatic morphogenesis and functional differentiation when associated with either embryonic urogenital sinus epithelium (UGE) or urothelium derived from adult urinary bladder (ABLE) and grown in male hosts. To determine the role of mesenchyme in prostatic acinar growth, homotypic tissue recombinants of 16-day-old embryonic mouse urogenital sinus mesenchyme and epithelium (UGM_mouse + UGE_mouse) were prepared in which the relative amounts of UGM to UGE were varied from approximately 0.1:1 to 100: 1. Recombinants were grown for 1 month in intact male hosts after which the amount of acinar growth was assessed by histological analysis and by determination of wet weight and DNA content. The latter criteria are valid measures of acinar content of histologically normal prostatic tissue because the rodent prostate is composed of greater than 80% ductalacinar tissue. From this analysis the amount of acinar growth was found to be determined by the amount of mesenchymal tissue and was independent of the amount of epithelium utilized to prepare the tissue recombinants. Similarly, the magnitude of growth in heterospecific rat-mouse tissue recombinants prepared with urogenital sinus epithelium or adult bladder epithelium (ABLE) and urogenital sinus mesenchyme (UGM_mouse + UGE_rat, UGM_mouse + ABLE_rat UGM_rat + UGE_mouse, or UGM_rat + ABLE_mouse) is determined by the source of UGM. Although the initial DNA content per UGM is comparable between mouse and rat, the overall acinar growth induced by UGM_rat is about 10-fold greater than that induced by UGM_mouse when recombined reciprocally with UGE_mouse and UGE_rat, respectively. These results suggest that UGM is of fundamental importance as a regulator of prostatic epithelial growth. The relevance of this finding to the development of human benign prostatic hyperplasia is discussed.     

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines

BACKGROUND: The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. METHODS: Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. RESULTS: As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. CONCLUSION: The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.     

Prostatic growth effects of rat urogenital sinus and human prostatic tissue in the rat

It is known that fetal urogenital sinus (UGS) implanted into one ventral lobe of an adult mouse prostate produces a significantly increased DNA content in that lobe. In our study, we attempted to determine whether that growth was of host or of graft tissue. UGS from fetal rats and ventral prostate from adult rats were implanted in various sites in syngeneic Fischer rats, alone or as a recombinant tissue complex. Human benign prostatic hyperplasia (BPH) tissue was implanted into the ventral prostate of adult, male, hooded nude rats to determine whether it stimulated prostatic growth. All UGS-implanted in situ ventral lobes enlarged. Mean DNA content of UGS and ventral prostate implanted together in the flank was not different from the mean DNA content of UGS and ventral prostate implanted separately in the flank (P = 0.69). Mean DNA content of in situ control ventral lobe and of UGS grown singly in the flank was less than that of a UGS-implanted in situ ventral lobe (P less than 0.01). Thus overgrowth in the UGS-implanted in situ ventral prostate was location-dependent, but which tissue(s) enlarged remains unclear. Implanted human BPH tissue did not stimulate rat prostatic growth.     

Ventral prostate predominant l, a novel mouse gene expressed exclusively in the prostate

BACKGROUND: Despite the region-specific nature of human prostate disease, there is a paucity of information regarding the molecular basis of prostate regionalization and patterning. To elucidate genetic mechanisms that underlie prostate growth and development, we investigated differential gene expression in mouse prostate lobes. METHODS: mRNA differential display analysis was used to identify differentially expressed genes during development of ventral, anterior, and dorsolateral prostate lobes. Differential gene expression was confirmed by Northern blot analysis and RT-PCR. RESULTS: A novel gene, Ventral prostate predominant1 (Vpp1) was identified. Vpp1 mRNA was evident in all lobes but accumulated predominantly in the ventral prostate, and was detected on postnatal day 7 through adulthood exclusively in the prostate gland. The steady-state level of Vpp1 mRNA decreased markedly in response to castration, suggesting androgen regulation of Vpp1 expression. Analysis of TRAMP tumors demonstrated a dramatic decrease in the level of Vpp1 mRNA. CONCLUSIONS: The spatial distribution and early postnatal onset of Vpp1 expression is consistent with a role for this gene in prostate regionalization. The absolute prostate specificity of Vpp1 expression may allow this gene to serve as a paradigm to study the molecular basis of gene expression that is restricted exclusively to the prostate gland.     

Altered expression of extracellular matrix and proteinases in Noble rat prostate gland after long-term treatment with sex steroids

BACKGROUND: Interactions between the epithelial cells and stromal tissues, which include the epithelial basement membrane, extracellular matrix, inducible factors, and various cell types, are believed to play a significant role in prostate gland carcinogenesis. Remodeling of extracellular matrix and degradation of basement membrane are the prerequisites for tumor cell invasion, and these changes are correlated with the expression of various proteinases. METHODS: The present study examined the alterations of epithelial basement membrane, extracellular matrix, and proteinase activities in the Noble rat prostate gland after long-term treatments with androgen and estrogen (T+DES or T+E(2) for 4-12 months) by histochemistry, immunohistochemistry, electron microscopy, and gelatin-gel zymography. RESULTS: After hormonal treatments, defects of epithelial basement membranes, such as focal disruption, diffuse staining and multilayering, were observed by histochemistry and immunohistochemistry in the dysplastic and neoplastic lesions induced in the lateral (LP) and ventral prostates (VP) but not in dorsal prostate (DP). An increase in the amount of extracellular matrix components, including hyaluronan (HA), chondroitin sulfate proteoglycan (CSPG) and tenascin, in the stroma of hormone-treated LP and VP was revealed by histochemistry and immunohistochemistry. Positive immunolabeling of matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) was detected in the fibromuscular layer surrounding the adenoma and adenocarcinoma induced in LP and VP after treatments with steroids for over 9-12 months. Zymography also detected an increase in activities of proteinases of apparent MW 120, 90, 86 and 68 kDa in the hormone-treated LP and VP, and these proteinases were characterized as metalloproteinases. In addition, two serine proteinases of MW 100 and 30 kDa were identified as being overexpressed in the hormone-treated LP and VP. Compared to LP and VP, there was no significant change in the proteinase activities in the hormone-treated DP. CONCLUSIONS: The present study demonstrated that the epithelial basement membrane and stromal extracellular matrix were altered in dysplastic and neoplastic Noble rat prostates. Since HA and CSPG (or their complexes) are highly anionic molecules, their increased accumulation in the altered prostatic stroma would tend to hydrate this tissue. This would create an environment more favorable for tumor growth and invasion. These morphological changes were also correlated with the concurrent increase in gelatinolytic proteinase activities induced in these prostates. The results suggest that the remodeling of the stromal tissue might play a role in the early stage of prostate carcinogenesis as shown in the Noble rat model.     

Extended culturing of androgen-responsive human primary epithelial prostate cell isolates by continuous treatment with interstitial collagenase

Continuous culturing of two distinct human prostate specimens in the presence of interstitial collagenase added directly to conventional medium resulted in the isolation and extended growth of primary epithelial prostate cell (PEPC) cultures from each. Both continued to proliferate substantially beyond the average time determined for analogous untreated epithelial prostate isolates. Both repeatedly stain positive for keratin and are characteristically epithelial in morphological appearance and growth model. Both express androgen receptor mRNA and stain positive for androgen receptors. PEPC-2 displays an androgen dose-dependent stimulation of cell proliferation, as well as specifically binding ³H-R1881. PEPC-1 exhibits a hypotetraploid karyotype with loss of the Y chromosome. PEPC-2 conserves a normal human ploidy, including the Y chromosome, although there is extensive random chromosome loss. Elimination of the collagenase from the medium resulted in decreased cellular proliferation and accumulation of stainable collagen in both PEPC cultures. Neither PEPC isolate produced tumors in male nude mice, whether injected alone, mixed with matrigel, or combined with prostate or bone fibroblastic cells. Prostate 30:7–19, 1997 © 1997 Wiley-Liss, Inc.     

Partial purification of a major type of rat prostatic growth factor: characterization as an epidermal growth factor-related mitogen

The dorsolateral prostate of rats contains a mitogen that shares several properties with epidermal growth factor (EGF), which was designated as prostatic EGF-related mitogen (PEM). PEM was purified about 2,100-fold using molecular-sieve and ion-exchange chromatography. Final preparation stimulated DNA synthesis in BALB/c 3T3 cells at a concentration as low as 1.5 ng/ml and competed with 125I-EGF for binding to cell surface receptors. PEM had a molecular weight of about 14,000 and an isoelectric point of about 4.5, being heat- and acid-stable but inactivated by dithiothreitol. The primary cultured rat dorsolateral prostate epithelial cells required EGF for maximum growth. Partially purified PEM fully substituted for EGF in the primary culture system at a concentration as low as 90 ng/ml. However, the activity of PEM was hardly suppressed by antimouse EGF antiserum. These findings suggest that PEM is a member of the EGF family but has a higher molecular weight (high molecular weight EGF).     

Increased expression of lymphocyte-derived cytokines in benign hyperplastic prostate tissue, identification of the producing cell types, and effect of differentially expressed cytokines on stromal cell proliferation

BACKGROUND: Benign prostatic hyperplasia (BPH) frequently exhibit infiltration of CD4 (+)/CD45RO (+) memory-T-lymphocytes. Expression and impact of lymphocyte-derived growth factors on prostatic stromal cell (PSC) growth were investigated. METHODS; Lymphokine synthesis in normal prostate tissues (n = 3), BPH-tissues (n = 13), BPH-derived T-cells (n = 6), BPH-derived epithelial cells (BPH-EC) (n = 5), normal prostate-derived (n = 3) and BPH-derived stromal cell lines (BPH-SC) (n = 6), and prostate cancer (CaP) lines (n = 3) was analyzed by RT-PCR and Southern-blotting. The effect of interleukin (IL)-2, -4, -7, and interferon-gamma (IFN-gamma) on normal and BPH-SC growth was investigated by (3)H-thymidine incorporation assays. RESULTS: All BPH-tissues and, to a lesser degree, normal prostates, expressed significant amounts of IFN-gamma mRNA. However, only BPH-tissues contained IL-2 and IL-4 mRNA (ratio: 10:13). BPH-T-cell lines were heterogeneous in composition and expressed significant amounts of IFN-gamma, IL-2, and IL-4 mRNA. Low level expression of these lymphokines was also observed in BPH-EC, CaP lines, and PSC lines. IL-2, -7 and IFN-gamma stimulated the proliferation of BPH-PSC lines but not that of normal PSC, while IL-4 inhibited BPH-PSC growth. CONCLUSIONS: Chronic inflammation may induce an increased growth pattern of fibromuscular tissue in BPH similar to that of wound healing.     

Prostate stem cells and benign prostatic hyperplasia

Pharmacological approaches are available to medically-managed patients with symptomatic BPH before surgical intervention is required. These include daily treatment with alpha-blockers and 5-alpha-reductase inhibitors alone or in combination. These medical approaches have two major problems. First, treatments are chronic and must be taken daily. Second, there are significant financial costs and quality of life issues for such chronic treatments. Is it possible to develop effective acute therapy for symptomatic BPH without the long-term androgen deprivation-induced side effects? Two seminal but rarely cited studies of Walsh [Peters, Walsh: N Engl J Med 317:599-604, 1987] and Coffey et al. [Sufrin et al.: Invest Urol 13:418-423, 1976], combined with the growing understanding of the stem cell organization of the prostate stromal (S) and epithelial (E) compartments and their reciprocal paracrine and autocrine interactions provides the rationale for an acute approach.The Walsh study documents that: (1) androgen deprivation disrupts the reciprocal interaction between the prostate S and E thereby decreasing the weight of both compartments and (2) once BPH develops, androgen deprivation does not decrease the number of stem cell units in either the S or E compartments since subsequent androgen restoration fully restores the enlarged gland. The Coffey study documents that acute androgen deprivation sensitizes S-E interactions to radiation induced disruptions so that following radiation, androgen restoration does not induce full gland regrowth. Therefore, effective therapy for symptomatic BPH should be achievable by acute treatment with reversible androgen deprivation for a limited period followed by a single dose of conformal external beam radiation before allowing the man to recovery his normal serum testosterone.     

Tissue interactions and prostatic growth: II. Morphological and biochemical characterization of adult mouse prostatic hyperplasia induced by fetal urogenital sinus implants

Current hypotheses regarding the causes of human benign prostatic hyperplasia have implicated both steroid hormone imbalance and tissue interactions. To examine the role of the latter we have further investigated the phenomenon of urogenital sinus-induced hyperplasia of the adult mouse ventral prostate. Urogenital sinuses (UGS) or purified urogenital mesenchyme (UGM) from C3H mice were implanted into the ventral prostates or coagulating glands of 50- to 90-day-old BALB/c-nu/nu hosts. The animals were sacrificed at 15, 30, and 180 days postimplantation to establish time dependence. Wet weight and DNA content were used as measures of net growth. Glucose phosphate isomerase (GPI) isozyme analysis was used to determine the relative contributions of C3H and BALB/c cells to the enlarged chimeric ventral prostates. It was determined that the induced growth is time-dependent and that the UGS induces 2- to 4-fold more growth than UGM. GPI analysis shows that UGM-induced growth was composed primarily of host-derived cells whereas the UGS is composed nearly equally of host- and implant-derived cells. Histologic analysis reveals that the UGS implants induce marked epithelial proliferation. The proliferating glands occur in clusters, and the epithelium within the glands appears cribriform. Foci of postobstructive cystic atrophy are also found. Remnants of the implanted UGS are still present even at 180 days postimplantation. UGM-induced growth is of a more subtle nature and appears morphologically similar to the sham-operated controls. In view of the morphologic similarity with human disease, as well as the time and hormonal dependence of UGS-induced ventral prostatic hyperplasia, this model represents the basis for a unified hypothesis regarding the roles of tissue interaction and hormonal milieu in human benign prostatic hyperplasia.     

Identification and characterization of PLZF as a prostatic androgen-responsive gene

BACKGROUND: Promyelocytic leukemia zinc finger protein (PLZF) was initially identified by virtue of its fusion with RARalpha as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients. PLZF has been reported to have pro-apoptotic and anti-proliferative activity both in vivo and in vitro. METHODS: Using a modified subtractive hybridization, we identified PLZF as an androgen-responsive gene in the rat ventral prostate. Northern blot and Western blot were used to characterize the regulation of PLZF by androgens in LNCaP cells. Stable transfections of PLZF in LNCaP cells were performed to assay the effect of PLZF overexpression on LNCaP cell proliferation. RESULTS: PLZF mRNA was transiently up-regulated by androgens in the regressed ventral prostate of castrated adult rat. PLZF was also up-regulated by androgens, at both mRNA and protein levels, in the androgen-responsive human prostate cancer cell line LNCaP. Androgen induction of PLZF mRNA was not inhibited by protein synthesis inhibitor cycloheximide but inhibited by androgen receptor antagonist bicalutamide, indicating that PLZF is a direct androgen-responsive gene. To study the functions of PLZF in androgen action, LNCaP sublines stably overexpressing PLZF were generated. PLZF overexpression inhibited LNCaP proliferation either in the presence or absence of androgen, which is consistent with the reported anti-proliferative activity of PLZF. CONCLUSIONS: The above observations indicate that PLZF is an androgen-responsive gene with anti-proliferative activity in prostate cancer cells.